DESCRIPTION: The long range goal of this application is to understand the cellular mechanisms that regulate the assembly of exocytotic release zones in chromaffin cells. These cells are used as a model system for understanding synaptic transmission. Much of the proposal stems from interesting new data of this group indicating that in chromaffin cells hotspots of calcium entry are colocalized with discrete exocytotic release zones. The application contains three specific aims. 1. To determine the types of voltage dependent channels that form the hotspots. By combining immunocytochemistry, PCR cloning, pulse laser imaging, patch clamp recording and specific calcium channel blockers, the investigator will attempt to determine which calcium channel type is specifically localized at the hotspots. 2. To determine the role of intracellular calcium channels in hotspot formation. The methods mentioned above will be used in conjunction with caged calcium and caged IP3 to determine the location of intracellular calcium release sites in relation to the hotspots. Intracellular calcium channel toxins will be used to determine the contribution of these channels to the formation of hotspots. 3. To determine whether a close association between docked vesicles and calcium channels determines the properties of a secretory response at the hotspots. Pulse laser imaging, caged compounds and amperometry will be used to determine calcium dependency. Latency distribution and sensitivity to calcium buffers of vesicle fusion at hotspots and away from them.